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1.
Scand J Immunol ; 91(1): e12805, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31267543

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by a mutation in the WAS gene that encodes the WAS protein (WASp); up to 5-10% of these patients develop inflammatory bowel disease (IBD). The mechanisms by which WASp deficiency causes IBD are unclear. Intestinal microbial dysbiosis and imbalances in host immune responses play important roles in the pathogenesis of polygenetic IBD; however, few studies have conducted detailed examination of the microbial alterations and their relationship with IBD in WAS. Here, we collected faecal samples from 19 children (all less than 2 years old) with WAS and samples from WASp-KO mice with IBD and subjected them to 16S ribosomal RNA sequencing. We found that microbial community richness and structure in WAS children were different from those in controls; WAS children revealed reduced microbial community richness and diversity. Relative abundance of Bacteroidetes and Verrucomicrobiain in WAS children was significantly lower, while that of Proteobacteria was markedly higher. WASp-KO mice revealed a significantly decreased abundance of Firmicutes. Faecal microbial dysbiosis caused by WASp deficiency is similar to that observed for polygenetic IBD, suggesting that WASp may play crucial function in microbial homoeostasis and that microbial dysbiosis may contribute to IBD in WAS. These microbial alterations may be useful targets for monitoring and therapeutically managing intestinal inflammation in WAS.


Assuntos
Disbiose , Fezes/microbiologia , Microbioma Gastrointestinal , Síndrome de Wiskott-Aldrich/etiologia , Animais , Biodiversidade , Biomarcadores , Estudos de Casos e Controles , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Lactente , Doenças Inflamatórias Intestinais/etiologia , Masculino , Metagenoma , Metagenômica/métodos , Camundongos , Camundongos Knockout , Mutação , RNA Ribossômico 16S/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/deficiência
2.
Nat Med ; 25(1): 130-140, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510251

RESUMO

In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-ß. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.


Assuntos
Linfoma de Células T/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Quinase do Linfoma Anaplásico/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Linfoma de Células T/enzimologia , Linfoma de Células T/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Ligação Proteica , Fator de Transcrição STAT3/metabolismo , Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína cdc42 de Ligação ao GTP/metabolismo
3.
J Cell Biol ; 216(12): 4073-4090, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29150539

RESUMO

The antimicrobial defense activity of neutrophils partly depends on their ability to form neutrophil extracellular traps (NETs), but the underlying mechanism controlling NET formation remains unclear. We demonstrate that inhibiting cytoskeletal dynamics with pharmacological agents or by genetic manipulation prevents the degranulation of neutrophils and mitochondrial DNA release required for NET formation. Wiskott-Aldrich syndrome protein-deficient neutrophils are unable to polymerize actin and exhibit a block in both degranulation and DNA release. Similarly, neutrophils with a genetic defect in NADPH oxidase fail to induce either actin and tubulin polymerization or NET formation on activation. Moreover, neutrophils deficient in glutaredoxin 1 (Grx1), an enzyme required for deglutathionylation of actin and tubulin, are unable to polymerize either cytoskeletal network and fail to degranulate or release DNA. Collectively, cytoskeletal dynamics are achieved as a balance between reactive oxygen species-regulated effects on polymerization and glutathionylation on the one hand and the Grx1-mediated deglutathionylation that is required for NET formation on the other.


Assuntos
Citoesqueleto/imunologia , Armadilhas Extracelulares/imunologia , Glutationa/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Actinas/genética , Actinas/imunologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Citoesqueleto/ultraestrutura , DNA Mitocondrial/imunologia , DNA Mitocondrial/metabolismo , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/imunologia , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteínas de Homeodomínio/imunologia , Humanos , Camundongos , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oxirredução , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
4.
Integr Biol (Camb) ; 9(8): 695-708, 2017 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-28678266

RESUMO

Dendritic cell migration to the T-cell-rich areas of the lymph node is essential for their ability to initiate the adaptive immune response. While it has been shown that the actin cytoskeleton is required for normal DC migration, the role of many of the individual cytoskeletal molecules is poorly understood. In this study, we investigated the contribution of the Arp2/3 complex binding protein, haematopoietic lineage cell-specific protein 1 (HS1), to DC migration and force generation. We quantified the random migration of HS1-/- DCs on 2D micro-contact printed surfaces and found that in the absence of HS1, DCs have greatly reduced motility and speed. This same reduction in motility was recapitulated when adding Arp2/3 complex inhibitor to WT DCs or using DCs deficient in WASP, an activator of Arp2/3 complex-dependent actin polymerization. We further investigated the importance of HS1 by measuring the traction forces of HS1-/- DCs on micropost array detectors (mPADs). In HS1 deficient DCs, there was a significant reduction in force generation (3.96 ± 0.40 nN per cell) compared to WT DCs (13.76 ± 0.84 nN per cell). Interestingly, the forces generated in DCs lacking WASP were only slightly reduced compared to WT DCs. Taken together, these findings show that HS1 and Arp2/3 complex-mediated actin polymerization are essential for the most efficient DC random migration and force generation.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Células Dendríticas/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Actinas/metabolismo , Animais , Bioengenharia , Fenômenos Biofísicos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos/deficiência , Fator Estimulador de Colônias de Granulócitos/genética , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/fisiologia
5.
Sci Rep ; 6: 30636, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27477778

RESUMO

To kill target cells, natural killer (NK) cells organize signaling from activating and inhibitory receptors to form a lytic synapse. Wiskott-Aldrich syndrome (WAS) patients have loss-of-function mutations in the actin regulator WASp and suffer from immunodeficiency with increased risk to develop lymphoreticular malignancies. NK cells from WAS patients fail to form lytic synapses, however, the functional outcome in vivo remains unknown. Here, we show that WASp KO NK cells had decreased capacity to degranulate and produce IFNγ upon NKp46 stimulation and this was associated with reduced capacity to kill MHC class I-deficient hematopoietic grafts. Pre-treatment of WASp KO NK cells with IL-2 ex vivo restored degranulation, IFNγ production, and killing of MHC class I negative hematopoietic grafts. Moreover, WASp KO mice controlled growth of A20 lymphoma cells that naturally produced IL-2. WASp KO NK cells showed increased expression of DNAM-1, LAG-3, and KLRG1, all receptors associated with cellular exhaustion and NK cell memory. NK cells isolated from WAS patient spleen cells showed increased expression of DNAM-1 and had low to negative expression of CD56, a phenotype associated with NK cells exhaustion. Finally, in a cohort of neuroblastoma patients we identified a strong correlation between WASp, IL-2, and patient survival.


Assuntos
Antineoplásicos/metabolismo , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Linfoma/imunologia , Microambiente Tumoral/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Antígeno CD56/análise , Degranulação Celular , Citotoxicidade Imunológica , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/química , Linfoma/mortalidade , Linfoma/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sobrevida
6.
Nat Commun ; 7: 12175, 2016 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-27425374

RESUMO

Wiskott-Aldrich syndrome (WAS) is caused by loss-of-function mutations in the WASp gene. Decreased cellular responses in WASp-deficient cells have been interpreted to mean that WASp directly regulates these responses in WASp-sufficient cells. Here, we identify an exception to this concept and show that WASp-deficient dendritic cells have increased activation of Rac2 that support cross-presentation to CD8(+) T cells. Using two different skin pathology models, WASp-deficient mice show an accumulation of dendritic cells in the skin and increased expansion of IFNγ-producing CD8(+) T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells leads to marked expansion of CD8(+) T cells at the expense of CD4(+) T cells. WASp-deficient dendritic cells induce increased cross-presentation to CD8(+) T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveals an intricate balance between activation of WASp and Rac2 signalling pathways in dendritic cells.


Assuntos
Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Deleção de Genes , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Proliferação de Células , Interferon gama/metabolismo , Leishmania major/fisiologia , Contagem de Linfócitos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/metabolismo , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
7.
Mol Ther ; 24(2): 342-353, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26502776

RESUMO

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Modelos Biológicos , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Antígenos Comuns de Leucócito/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo
8.
J Clin Invest ; 125(10): 3941-51, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26368308

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.


Assuntos
Linfócitos B/imunologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Tolerância Imunológica , Proteína da Síndrome de Wiskott-Aldrich/uso terapêutico , Síndrome de Wiskott-Aldrich/terapia , Adulto , Sequência de Aminoácidos , Medula Óssea/patologia , Criança , Pré-Escolar , Deleção Clonal , Células Clonais/imunologia , Humanos , Lentivirus/genética , Masculino , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão , Linfócitos T Reguladores/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
9.
Pediatr Blood Cancer ; 62(9): 1601-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25931402

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by mutations of the WAS gene. The genotype-phenotype association of WAS and XLT have not been fully elucidated. Here, we established the largest database of WAS in China to further determine the potential correlation between genotype and phenotype and long-term outcome. PROCEDURES: We collected clinical data of 81 WAS/XLT patients, analyzed mutations of WAS gene at the genomic DNA and transcriptional/translational levels, and quantified three different patterns of WAS protein (WASp) expression in PBMCs by flow cytometry. RESULTS: There were 60 unique mutations identified, including 20 novel mutations and eight hotspots, from 75 unrelated families with a total of 81 affected members. Nearly all the patients with XLT had missense mutations and were WASp-positive in the peripheral cells, while only half of the patients with missense mutations exhibited the XLT phenotype and detectable WASp. In contrast, patients with nonsense mutations, deletions, insertions, and complex mutations were WASp-negative and developed the classic WAS phenotype. An equal number of patients with splice anomalies were either WASp-positive or WASp-negative. Long-term survival rates were lower in WASp-negative patients compared to WASp-positive patients. CONCLUSIONS: The clinical phenotype of classic WAS or milder XLT and long-term outcome are potentially influenced by the effect of these defects on gene transcription and translation. Patients with missense mutations allowing expression of mutated WASp and those with splice anomalies, which result in generation of multiple products, including normal WASp, present the attenuated XLT phenotype and show better prognosis.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Trombocitopenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Idade de Início , Pré-Escolar , China/epidemiologia , Análise Mutacional de DNA , Bases de Dados Genéticas , Doenças em Gêmeos/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/epidemiologia , Genótipo , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Biossíntese de Proteínas , Taxa de Sobrevida , Trombocitopenia/epidemiologia , Transcrição Genética , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/epidemiologia , Síndrome de Wiskott-Aldrich/terapia , Proteína da Síndrome de Wiskott-Aldrich/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/deficiência
10.
Scand J Immunol ; 81(6): 483-93, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25728049

RESUMO

The Wiskott-Aldrich syndrome protein (WASp) is an important regulator of the actin cytoskeleton and is required for immune cell function. WASp deficiency causes a marked reduction in major mature peripheral B cell subsets, particularly marginal zone (MZ) B cells. We hypothesized that WASp deficiency may also lead to a reduction of regulatory B cells (known as B10 cells) belonging to a novel subset of B cells. And in consideration of the key role of B10 cells play in maintaining peripheral tolerance, we conjectured that a deficit of these cells could contribute to the autoimmunity in patients with Wiskott-Aldrich syndrome (WAS). The effects of WASp deficiency on B10 cells have been reported by only one group, which used an antigen-induced arthritis model. To add more information, we measured the percentage of B10 cells, regulatory T cells (Tregs) and Th1 cells in WASp knockout (WASp KO) mice. We also measured the percentage of B10 cells in patients with WAS by flow cytometry. Importantly, we used the non-induced autoimmune WASp KO mouse model to investigate the association between B10 cell frequency and the Treg/Th1 balance. We found that the percentage of B10 cells was reduced in both mice (steady state and inflammatory state) and in humans and that the lower B10 population correlated with an imbalance in the Treg/Th1 ratio in old WASp KO mice with autoimmune colitis. These findings suggest that WASp plays a crucial role in B10 cell development and that WASp-deficient B10 cells may contribute to autoimmunity in WAS.


Assuntos
Linfócitos B Reguladores/imunologia , Interleucina-10/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos B Reguladores/metabolismo , Criança , Pré-Escolar , Colite/genética , Colite/imunologia , Colite/metabolismo , Citometria de Fluxo , Humanos , Lactente , Interleucina-10/metabolismo , Contagem de Linfócitos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
11.
Eur J Haematol ; 95(1): 93-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25082437

RESUMO

Deletion mutations of WAS are relatively rare and the precise localization of large deletions in the genome has rarely been described in previous studies. We report here a 5-month-old boy with a large deletion mutation in WAS that completely abolished protein expression. To localize the deletion, a 2816-bp-length sequence that spans between exons 9 and 12 was amplified. PCR amplification of the patient's sample revealed a single band of about 1 kb in contrast to the 2816-bp-amplicon in the control. Genomic DNA sequencing of the patient revealed a 1595-bp-deletion and an adenine insertion (g.5247_6841del1595insA). This large deletion of WAS resulted in partial loss of exon 10 and intron 11, and a complete loss of intron 10 and exon 11.


Assuntos
Éxons , Mutação INDEL , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/genética , Sequência de Bases , Humanos , Lactente , Íntrons , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência
12.
Eur J Immunol ; 44(4): 1039-45, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24338698

RESUMO

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor-prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was(-/-) NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was(-/-) mice that had received WT NK cells as compared with mice bearing Was(-/-) NK cells. Furthermore, we demonstrated that Was(-/-) DCs were less efficient in inducing NK-cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.


Assuntos
Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Animais , Transplante de Medula Óssea , Linhagem Celular Tumoral , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Estimativa de Kaplan-Meier , Células Matadoras Naturais/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carga Tumoral/genética , Carga Tumoral/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
13.
J Autoimmun ; 50: 42-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24369837

RESUMO

Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21(low) B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.


Assuntos
Autoimunidade , Linfócitos B/imunologia , Suscetibilidade a Doenças/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Síndrome de Wiskott-Aldrich/imunologia , Fator Ativador de Células B/sangue , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/patologia , Diferenciação Celular , Movimento Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Expressão Gênica , Homeostase/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Memória Imunológica , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
14.
Haematologica ; 98(8): 1300-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23445877

RESUMO

The Wiskott-Aldrich syndrome protein is an essential cytoskeleton regulator found in cells of the hematopoietic lineage and controls the motility of leukocytes. The impact of WAS gene deficiency on the mobilization of hematopoietic progenitor/stem cells in circulation has remained unexplored but information would be pertinent in the context of autologous gene therapy of Wiskott-Aldrich syndrome. The response to granulocyte-colony stimulating factor mobilization was investigated in a murine WAS knock-out model of the disease, by measuring hematologic parameters, circulation and engraftment of hematopoietic progenitor/stem cells. In the steady-state, adult WAS knock-out mice have B-cell lymphopenia, marked neutrophilia, increased counts of circulating hematopoietic progenitor cells and splenomegaly, presumably caused by the retention of hematopoietic progenitor cells due to high levels of splenic CXCL12. In spite of these anomalies, the administration of granulocyte-colony-stimulating factor mobilizes progenitor/stem cells in WAS knock-out mice to the same level and with the same kinetics as in wild-type control mice. Mobilized peripheral blood cells from WAS knock-out mice can be transduced and are able to engraft into lethally-irradiated hosts reconstituting multiple lineages of cells and providing more effective radio-protection than mobilized cells from wild-type control mice. Surprisingly, the homing and the peripheral blood recovery of B lymphocytes was influenced by the background of the host. Thus, in the absence of Wiskott-Aldrich syndrome protein, effective mobilization is achieved but partial correction may occur as a result of an abnormal hematopoietic environment.


Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/terapia , Animais , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Proteínas Recombinantes/farmacologia
15.
Dis Model Mech ; 6(2): 544-54, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23324327

RESUMO

Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFß and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS patients and as a cellular model to study new therapeutic strategies.


Assuntos
Desoxirribonucleases/metabolismo , Técnicas de Inativação de Genes , Modelos Biológicos , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Dedos de Zinco , Actinas/metabolismo , Animais , Apoptose/genética , Adesão Celular/genética , Diferenciação Celular/genética , Reparo do DNA , Regulação para Baixo/genética , Marcação de Genes , Recombinação Homóloga/genética , Humanos , Células K562 , Leucossialina/metabolismo , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
16.
J Exp Med ; 210(2): 355-74, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23337808

RESUMO

Mutations in Wiskott-Aldrich syndrome (WAS) protein (WASp), a regulator of actin dynamics in hematopoietic cells, cause WAS, an X-linked primary immunodeficiency characterized by recurrent infections and a marked predisposition to develop autoimmune disorders. The mechanisms that link actin alterations to the autoimmune phenotype are still poorly understood. We show that chronic activation of plasmacytoid dendritic cells (pDCs) and elevated type-I interferon (IFN) levels play a role in WAS autoimmunity. WAS patients display increased expression of type-I IFN genes and their inducible targets, alteration in pDCs numbers, and hyperresponsiveness to TLR9. Importantly, ablating IFN-I signaling in WASp null mice rescued chronic activation of conventional DCs, splenomegaly, and colitis. Using WASp-deficient mice, we demonstrated that WASp null pDCs are intrinsically more responsive to multimeric agonist of TLR9 and constitutively secrete type-I IFN but become progressively tolerant to further stimulation. By acute silencing of WASp and actin inhibitors, we show that WASp-mediated actin polymerization controls intracellular trafficking and compartmentalization of TLR9 ligands in pDCs restraining exaggerated activation of the TLR9-IFN-α pathway. Together, these data highlight the role of actin dynamics in pDC innate functions and imply the pDC-IFN-α axis as a player in the onset of autoimmune phenomena in WAS disease.


Assuntos
Actinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon Tipo I/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/antagonistas & inibidores , Animais , Autoimunidade , Sequência de Bases , Células Dendríticas/patologia , Modelos Animais de Doenças , Endocitose , Feminino , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon-alfa/biossíntese , Interferon-alfa/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
17.
Platelets ; 24(4): 288-96, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22812495

RESUMO

The thrombocytopenia of Wiskott-Aldrich syndrome (WAS) is thought to be due to both reduced platelet production and accelerated platelet consumption. We have previously demonstrated that platelets from WASP-deficient mice are consumed more rapidly in vivo than are WT platelets, and that opsonization accelerates their uptake by bone marrow- derived macrophages more than it does that of WT platelets. Here we asked whether platelets from WAS patients show similar features. We show that ex vivo phagocytosis by activated THP-1 cells of DIO-labeled platelets from a series of WAS or XLT patients is increased in comparison to that of normal control platelets. Using a numerical analysis method, we distinguish this effect from a concurrent effect on the amount of detectable fluorescent signal transferred to the macrophage per phagocytosed platelet. We show that the latter quantity is reduced by platelet WASP deficiency, as might be expected if the fluorescence transferred from these smaller platelets is more rapidly quenched. We are unable to detect a differential effect of opsonization with anti-CD61 antibody on the uptake of WASP(-) vs. WT platelets. However, the high probability of phagocytosis per adsorbed WASP(-) platelet could limit the sensitivity of the assay in this case. We also see no effect of sera from WAS patients on the uptake of normal control platelets, suggesting that in vivo opsonization is not the cause of increased uptake of WASP(-) platelets. Finally, we show little, if any, increase in the reticulated platelet fraction in WAS patients, suggesting that impaired production of reticulated platelets contributes to the thrombocytopenia. Our findings suggest that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. They also demonstrate the feasibility of routinely performing functional assays of phagocytosis of small numbers of platelets obtained at remote locations, a method which should be applicable to the study of other types of thrombocytopenia such as ITP.


Assuntos
Plaquetas/imunologia , Fagocitose/imunologia , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/imunologia , Plaquetas/metabolismo , Linhagem Celular , Humanos , Contagem de Plaquetas , Proteína da Síndrome de Wiskott-Aldrich/deficiência
18.
J Allergy Clin Immunol ; 131(3): 815-24, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23141740

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by absence of Wiskott-Aldrich syndrome protein (WASP) expression, resulting in defective function of many immune cell lineages and susceptibility to severe bacterial, viral, and fungal infections. Despite a significant proportion of patients with WAS having recurrent viral infections, surprisingly little is known about the effects of WASP deficiency on antiviral immunity. OBJECTIVE: We sought to evaluate the antiviral immune response in patients with WASP deficiency in vivo. METHODS: Viral clearance and associated immunopathology were measured after infection of WASP-deficient (WAS KO) mice with lymphocytic choriomeningitis virus (LCMV). Induction of antiviral CD8(+) T-cell immunity and cytotoxicity was documented in WAS KO mice by means of temporal enumeration of total and antigen-specific T-cell numbers. Type I interferon (IFN-I) production was measured in serum in response to LCMV challenge and characterized in vivo by using IFN-I reporter mice crossed with WAS KO mice. RESULTS: WAS KO mice showed reduced viral clearance and enhanced immunopathology during LCMV infection. This was attributed to both an intrinsic CD8(+) T-cell defect and defective priming of CD8(+) T cells by dendritic cells (DCs). IFN-I production by WAS KO DCs was reduced both in vivo and in vitro. CONCLUSIONS: These studies use a well-characterized model of persistence-prone viral infection to reveal a critical deficiency of CD8(+) T-cell responses in murine WASP deficiency, in which abrogated production of IFN-I by DCs might play an important contributory role. These findings might help us to understand the immunodeficiency of WAS.


Assuntos
Infecções por Arenaviridae/imunologia , Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Infecções por Rhabdoviridae/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Humanos , Vírus da Coriomeningite Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vírus da Estomatite Vesicular Indiana , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
19.
Blood ; 121(1): 72-84, 2013 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-23160469

RESUMO

Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.


Assuntos
Células Dendríticas/patologia , Mutação de Sentido Incorreto , Proteína da Síndrome de Wiskott-Aldrich/genética , Actinas/metabolismo , Animais , Biopolímeros , Proteínas de Transporte/metabolismo , Movimento Celular , Células Cultivadas , Proteínas do Citoesqueleto , Células Dendríticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Polimerização , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Terciária de Proteína , Pseudópodes/patologia , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Organismos Livres de Patógenos Específicos , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/fisiologia
20.
Gastroenterology ; 143(3): 719-729.e2, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22710191

RESUMO

BACKGROUND & AIMS: Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich syndrome protein (WASP), a hematopoietic cell-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. METHODS: Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2-deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. RESULTS: Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared with RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin-10. Administration of interleukin-10 prevented induction of colitis following transfer of T cells into WRDKO mice. CONCLUSIONS: Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of interleukin-10, and homeostasis of regulatory T cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Colo/imunologia , Imunidade Inata , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Proliferação de Células , Células Cultivadas , Colite/genética , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica , Cadeias alfa de Integrinas/metabolismo , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ativação Linfocitária , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Quimeras de Transplante , Proteína da Síndrome de Wiskott-Aldrich/genética
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